Enzymatic Synthesis Of Non-protein Amino Acids And Their Use As Intermediates Involved In The Synthesis Of New Drugs

O-acetylserine sulfhydrylase A (OASS) is an important enzyme in the assimilationpathway of sulfate and cysteine synthesis. It presents as an enzyme complex form togetherwith serine acetyltransferase (SAT) in the synthesis of cysteine, usually called cysteinesynthase. L-Cysteine biosynthesis in microorganisms and higher plants is catalyzed in twosteps. The first step is carried out by the enzyme of serine acetyltransferase (SAT; EC2.3.1.30), which catalyzes the formation of O-acetyl-L-serine (OAS) from L-serine and acetylCoA, while the second step is carried out by O-acetylserine sulfhydrylase-A (OASS-A; EC2.5.1.47; O-acetylserine(thiol)-lyase), which catalyzes the formation of L-cysteine from OASand sulfide. OASS is able to utilize various nucleophiles and mercaptans to synthesizenon-protein amino acids by replacing sulfide substrate in the second step.Non-protein amino acids are other ones except for the20amino acids found in proteinsexisting in nature. For rich diversity of the peptide chain and breakthrough the barrier ofnatural amino acids, non-protein amino acids are provided as base materials in artificialsynthesis of protein. They are also attractive candidates, for using as building blocks of thesynthesis of active pharmaceutical ingredients that contain chiral centers and as substancesthat exhibit a variety of biological activities. Non-protein amino acids are widely used inresearch and medicine synthesis as the key intermediates.Full-length of cysK was972bp amplified by polymerase chain reaction (PCR) utilizingEscherichia coli (E. coli) DH5α genomic DNA as template and ligated with pET-22b(+)expression vector to construct prokaryotic expression plasmid. The recombinant wastransformed into competent E. coli BL21for protein expression with IPTG induction.Recombinant protein was purified with Ni2+-resin affinity chromatography and identified bySDS-PAGE. Experimental results showed that the prokaryotic expression vector for OASS-Awas successfully constructed and fusion protein was expressed at a high level with IPTGinduction. Enzyme activity was assayed for recombinant protein OASS-with O-acetyl-serineas substrate. The unit of enzyme activity of OASS-A was750U/mg in the purifiedrecombinant protein and400U/mg in the crude enzyme extract. Non-protein amino acidS-phenyl-L-cysteine (S-P-C) was effectively synthesized from substrate O-acetyl-serine andthiophenol, catalyzed by recombinant protein OASS-A. The purity and chemical structure ofthe synthesized compound were determined by HPLC and1H NMR techniques. Highly efficient and effective synthesis mode of non-protein amino acids was established throughbiocatalytic method of recombinant enzyme. According to the principles of drug stitching,S-P-C was coupled with effective groups of active compounds asiatic acid and deoxycholicacid. Inhibition of cancer cells proliferation was preliminarily tested by MTT assay. Theresults of S-P-C derivatives showed certain degree of antiproliferative effects.